All referenced patents and applications and publications are incorporated herein by reference in their entirety. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
The measurement of the optical density of a fluid that contains optical absorbers or scatters can yield valuable information about the sample, such as the quantity of the optical absorber or quantity of scatterers. Typically, optical density is measured by removing a sample of the fluid, placing it in a sample container, possibly diluting the sample, and measuring the optical transmission through the sample using instruments such as a spectrophotometer. In addition, the optical transmission through a blank, or reference sample is measured and optical density is then computed as −10*log 10(Tsample/Tblank). Measurement of transmission through a blank or reference sample is intended to cancel out sources of transmission reduction that are not intrinsic to the sample.
It is, however, desirable to perform optical density measurements online and in-situ without removing the sample, when sample removal is inconvenient, risky, or impossible. Examples include measurement of optical density in miniature, or microfluidic devices where there is not enough fluid to sample, including microfluidic bioreactors where it is desirable to monitor the cell density, in stirred tank bioreactors where sample removal increases contamination risk, or at the bottom of the ocean where sample retrieval is difficult.
In-situ optical density measurements are subject to errors because reference sample measurement is difficult. One method is to perform a reference measurement at the beginning of the experiment allowing changes in the optical density from a starting point to be recorded. This method suffers from error due to variations in the intensity of the light source and fouling of optical surfaces which cause a reduction in transmission that is not related to the optical density of the fluid. Another approach is to perform optical density measurements using two different path lengths. Using this approach, the transmission through the shorter path length serves as the reference for the transmission through the longer path length and the computed optical density refers to the optical density of the fluid in the difference between the path lengths. There are multiple methods for implementing multiple path lengths. One is to use two fixed, independent optical paths through the fluid. A problem with this approach is an error is introduced if the optical fouling on the surfaces of the different optical paths is not the same. Another is to introduce a transparent material into the optical path, as taught in U.S. Pat. No. 7,826,050, after a first measurement, thereby reducing the proportion of the optical path through the fluid, and then taking a reference measurement. A problem with this approach is that the reference measurement with the transparent material has additional optical surfaces that can be fouled when compared to the optical path without the transparent material. Yet another approach is to physically move the optical surfaces to change the optical path length as taught in U.S. Pat. No. 4,786,171, U.S. Pat. No. 5,168,367, U.S. Pat. No. 5,268,736, and U.S. Pat. No. 5,303,036. This method reduces the chances of error, however it is the most mechanically complicated to implement, requiring a sliding seal and mechanical means to change the optical path length.
Another source of interference that is more difficult to remove is, light scattering from gas bubbles which also reduce transmission in a way not related to the optical density of the fluid. Gas bubbles are common in systems where gas is a reactant and is supplied to the vessel in small gas bubbles. In addition, for certain biological or chemical processes, gas bubbles can be generated by the reaction.
Thus, there remains a considerable need for devices and methods to measure optical density in-situ that minimize errors due to optical fouling and gas bubbles.